Abstract

We hypothesized that cystine/glutamate transporters (xCTs) might be critical regulators of ambient extracellular glutamate levels in the nervous system and that misregulation of this glutamate pool might have important neurophysiological and/or behavioral consequences. To test this idea, we identified and functionally characterized a novel Drosophila xCT gene, which we subsequently named "genderblind" (gb). Genderblind is expressed in a previously overlooked subset of peripheral and central glia. Genetic elimination of gb causes a 50% reduction in extracellular glutamate concentration, demonstrating that xCT transporters are important regulators of extracellular glutamate. Consistent with previous studies showing that extracellular glutamate regulates postsynaptic glutamate receptor clustering, gb mutants show a large (200-300%) increase in the number of postsynaptic glutamate receptors. This increase in postsynaptic receptor abundance is not accompanied by other obvious synaptic changes and is completely rescued when synapses are cultured in wild-type levels of glutamate. Additional in situ pharmacology suggests that glutamate-mediated suppression of glutamate receptor clustering depends on receptor desensitization. Together, our results suggest that (1) xCT transporters are critical for regulation of ambient extracellular glutamate in vivo; (2) ambient extracellular glutamate maintains some receptors constitutively desensitized in vivo; and (3) constitutive desensitization of ionotropic glutamate receptors suppresses their ability to cluster at synapses.

genderblind mutants show an increase in postsynaptic glutamate receptor immunoreactivity. A, Relative concentration of glutamate in larval hemolymph, in gb mutants and precise-excision controls. gb[KG07905] mutants represent animals homozygous for a transposon insertion in the first exon of the gb gene (A, top). Precise-excision animals represent animals homozygous for the same chromosome after precise excision of the transposon (confirmed by sequencing of gb). n for each genotype equals 10 independently analyzed samples derived from 50 larvae. To control for variability in hemolymph extraction and/or evaporation, we normalized glutamate concentration measured in each sample by dividing it by the concentration of free phenylalanine in the same sample (see Materials and Methods). B,gb mutants show large increases in relative synaptic GluRIIA immunofluorescence, compared with controls (n = 6 per genotype). This large increase in GluR immunoreactivity is rescued when semi-intact gb and GOT mutants are cultured for 24 h in a physiologically normal (2 mM) hemolymph glutamate concentration. C, Portions of representative confocal micrographs showing larval NMJs on ventral longitudinal muscles 6/7, stained with anti-GluRIIA antibodies (green) and the neuronal membrane marker anti-HRP (magenta). White indicates overlap. Error bars represent SEM.

Time course of glutamate-dependent changes in postsynaptic glutamate receptor abundance. A, Synaptic GluRIIA immunoreactivity after a semi-intact neuromuscular preparation is cultured in glutamate for varying amounts of time and then immediately fixed and stained. B, Changes in GluRIIA immunoreactivity after 24 h when preparations are kept in 10 mM glutamate for the entire 24 h (first bar), kept in 10 mM glutamate for 12 h and then 0 mM glutamate for the next 12 h (second bar), or kept in 0 mM glutamate for the entire 24 h (third bar). C, Glutamate-dependent changes in postsynaptic GluRIIB immunoreactivity, as in A. D, Changes in GluRIIB immunoreactivity, as in B (n = 5–21 animals per time point). Error bars represent SEM.